Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for multiple processes within the eukaryotic cell, including membrane transport and neurotransmitter secretion. How the V-ATPase is regulated, e.g. by an accessory subunit, remains elusive. Here we explored the role of the neuroendocrine V-ATPase accessory subunit Ac45 via its transgenic expression specifically in the Xenopus intermediate pituitary melanotrope cell model. The Ac45-transgene product did not affect the levels of the prohormone proopiomelanocortin nor of V-ATPase subunits, but rather caused an accumulation of the V-ATPase at the plasma membrane. Furthermore, a higher abundance of secretory granules, protrusions of the plasma membrane and an increased Ca(2+)-dependent secretion efficiency were observed in the Ac45-transgenic cells. We conclude that in neuroendocrine cells Ac45 guides the V-ATPase through the secretory pathway, thereby regulating the V-ATPase-mediated process of Ca(2+)-dependent peptide secretion.     

Targeted disruption of the mouse gene encoding the V-ATPase accessory subunit Ac45

Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptormediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H + -ATPase (V-ATPase).How the activity of this multisubunit enzyme is regulated remains tobe established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function ofAc45, wedisrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (- /Y) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affectthe normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.     

Targeted disruption of the mouse gene encoding the V-ATPase accessory subunit Ac45

Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptor-mediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H(+)-ATPase (V-ATPase). How the activity of this multisubunit enzyme is regulated remains to be established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function of Ac45, we disrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (-IY) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affect the normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.     

Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary.

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.     

Versatile roles of V-ATPases accessory subunit Ac45 in osteoclast formation and function

Vacuolar-type H(+)-ATPases (V-ATPases) are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-Flox(Neo) mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function.     

Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.     

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H⁺)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.     

Architectural editing: determining the fate of newly synthesized membrane proteins

Many integral membrane proteins exist on the plasma membrane as part of multicomponent complexes. In addition to correctly transporting newly synthesized proteins from their site of synthesis in the endoplasmic reticulum to the plasma membrane, the cell must possess mechanisms to ensure that the complexes expressed on the cell surface are accurately assembled. The cell appears to accomplish this feat by superimposing a set of constraints on the newly synthesized membrane proteins whereby the structure and state of assembly of the protein determine its intracellular fate. These processes impose a dramatic level of post-translational regulation on the expression of surface membrane protein complexes. By and large, the cell uses these mechanisms to dispose of, or "edit out," newly synthesized proteins that are not correctly assembled or folded. This review will describe current views of the processes of architectural editing, with an emphasis on the regulation of cell surface expression of the multicomponent T-cell antigen receptor complex.     

Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas

Pancreatic lobules from fasted rats secrete pulse-labeled proteins in two phases comprising 15 and 85% of basal output, respectively. The first (0-6.5 h) is initially (less than or equal to 0.5 h) unstimulated by secretagogues, probably represents vesicular traffic of Golgi and post-Golgi origin (including condensing vaculoles/immature granules), and notably contains two groups of polypeptides with distinct release rates: zymogens (t1/2 approximately 2.4 h) and minor nonzymogens plus one unique zymogen (t1/2 approximately 1 h). The second phase (peak at 9-10 h) is stimulable, probably represents basal granule exocytosis (t1/2 approximately 5 h), and contains zymogens exclusively. Newly synthesized proteins released in both phases appear asynchronously, reiterating their asynchronous transport through intracellular compartments. Zymogens in both phases are secreted apically. The sorting of first from second phase zymogen release does not appear to be carrier-mediated, although the sorting of zymogens from other secretory proteins may use this process. Finally, data are presented that suggest that both secretory phases are subject to physiologic regulation.     

Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0) and the cytoplasmic V(1). Here we found that the ratio of membrane associated V(1)/Vo varies along the endocytic pathway, the relative abundance of V(1) being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.     

Intracellular trafficking pathway of newly synthesized CD1b molecules

The intracellular trafficking of major histocompatibility complex (MHC) class I and class II molecules has evolved to support their function in peptide antigen presentation optimally. We have analyzed the intracellular trafficking of newly synthesized human CD1b, a lipid antigen-presenting molecule, to understand how this relates to its antigen-presenting function. Nascent CD1b was transported rapidly to the cell surface after leaving the Golgi, and then entered the endocytic system by internalization via AP-2-dependent sorting at the plasma membrane. A second sorting event, possibly involving AP-3 complexes, led to prominent accumulation of CD1b in MHC class II compartments (MIICs). Functional studies demonstrated the importance of nascent CD1b for the efficient presentation of a foreign lipid antigen. Therefore, the intracellular trafficking of nascent CD1b via the cell surface to reach MIICs may allow the efficient sampling of lipid antigens present in endocytic compartments.     

Association of newly synthesized pro-opiomelanocortin with secretory granule membranes in pituitary pars intermedia cells

The prohormone, pro-opiomelanocortin (POMC) is synthesized on ribosomes, subsequently routed to the Golgi apparatus and finally packaged into secretory granules where it is processed to various biologically active hormones (alpha-melanotropin, adrenocorticotropin, beta-endorphin and beta-lipotropin). We report here that in frog and mouse pars intermedia cells, newly synthesized [3H]Arg-labeled POMC is associated with the secretory granule membrane prior to processing. This association with the secretory granule membrane may be related to the intracellular transport and packaging of POMC and/or the facilitation of processing of the prohormone within the organelle.     

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.     

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.     

The subcellular localization of newly synthesized cytochrome b5.

The fate of newly synthesized cytochrome b₅ was studied in rat hepatocytes. Using an antibody specific for microsomal cytochrome b₅, we found newly synthesized microsomal cytochrome b₅ in both mitochondria and a mitochondria associated membrane fraction as well as in microsomes. Newly synthesized cytochrome b₅ was quickly removed from the site of synthesis on free ribosomes and inserted into membranes at random. No migration of newly synthesized cytochrome b₅ between cellular compartments was observed and therefore the assembly of the apoprotein with the heme moiety is apparently not taking place in any particular cellular compartment.     

Dimer formation of subunit G of the yeast V-ATPase

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V(1) and V(O) sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28+/-2 kDa, indicating that this protein is dimeric. With a radius of gyration (R(g)) and a maximum size (D(max)) of 2.7+/-0.2 nm and 8.0+/-0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G(38-144) form (the carboxyl-terminus) was expressed and purified. G(38-144) is homogeneous, with a molecular mass of approximately 12+/-3 kDa, indicating a monomeric form in solution.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

The complete general secretory pathway in gram-negative bacteria.

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.